Comparative genomic hybridization array method for preimplantation genetic screening

ABSTRACT

A method for determining the presence of a copy number imbalance in genomic DNA of a test sample is provided. The method can separately measure hybridization of a single test sample to a first hybridization array and hybridization of a plurality of reference samples to a plurality of other, respective test arrays. A determination of copy number can be based on the best fit reference array, relative to the test array. The best fit can be determined based on the closest or most similar signal-to-noise ratio of the measured signals.

FIELD

The present teachings relates to methods for detecting geneticabnormalities within the cells of an embryo, oocyte, polar body, orassociated biopsy.

BACKGROUND

Within the field of IVF (in-vitro fertilization) it is desirable toidentify the number and complement of chromosomes within the cells of anembryo prior to implantation. There is increasing evidence that one ofthe most important factors influencing embryo viability is chromosomeimbalance, including copy number gain/loss and whole chromosomeaneuploidy (abnormal number of chromosomes).

Current methods for testing first involve isolation of the geneticmaterial which is representative of the embryo for testing. Samplescurrently used in the analysis of aneuploidy are a polar body biopsyassociated with the oocyte, a single cell from blastomere biopsy(associated with the day 3 embryo), or trophoectoderm biopsy (associatedwith the day 5 embryo, or blastocyst). In some cases, however, samplestaken at other or multiple points in the process prove more effective.The polar body or cell(s) are then tested via a choice of methods todetect copy number imbalance. For the purposes of the presentapplication, such testing methods will be referred to as preimplantationgenetic screening (PGS), although the term PGD is often encountered inthe literature. The term PGS shall also include testing of polar bodiesto access oocyte quality, for example, to enable informed egg banking.

Comparative genomic hybridization (CGH) is a technique that has beenemployed to detect the presence and identify the location of amplifiedor deleted sequences in genomic DNA, corresponding to so-called changesin copy number. Typically, genomic DNA is isolated from normal referencecells, as well as from test cells. The two nucleic acid samples aredifferentially labeled and then hybridized in-situ to metaphasechromosomes of a reference cell. The repetitive sequences in both thereference and test DNAs are either removed or their hybridizationcapacity is reduced by some means. Chromosomal regions in the test cellswhich are at increased or decreased copy number can be identified bydetecting regions where the ratio of signal from the two DNAs isaltered. The detection of such regions of copy number change can be ofparticular importance in the diagnosis of genetic disorders.

Metaphase CGH, as described above, has also been applied to and has theability to screen all chromosomes for abnormalities. For CGH analysis tobe applied to a PGS context, amplification of the entire genome isrequired to increase the quantity of DNA from a single cell (5-10 pg) tolevels suitable for metaphase CGH (1 μg) prior to analysis. Commonlyused methods for amplification include DOP-PCR (Telenius et al., 1992)or more recently whole-genome amplification kits such as, GENOMEPLEX(Rubicon genomics) and REPLI-G (Qiagen). The main problem with usingmetaphase CGH in a clinical setting is that it can take around 4 days tocomplete, which is not compatible with the time frame required for thepre-implantation of embryos in IVF, without the freezing of embryos andimplantation occurring in the following cycle. In addition, the methodis technically challenging and requires high levels of expertise tocarry out and analyze. These difficulties have limited the widespreaduse of metaphase CGH in PGS.

Pinkel et al. in 1998 and 2003 disclosed the technique which has becomewidely known as array comparative genomic hybridization, hereafterreferred to as arrayCGH. In 1998, Solinas-Toldo et al. described asimilar “Matrix-based comparative genomic hybridization” approach.

The arrayCGH technique relies on similar assay principles to CGH withregard to exploiting the binding specificity of double stranded DNA. InarrayCGH, the metaphase chromosomes of a reference cell are replacedwith a collection of potentially thousands of solid-support-boundunlabelled target nucleic acids (probes), for example, an array ofclones which have been mapped to chromosomal locations. ArrayCGH is thusa class of comparative techniques for the high throughput detection ofdifferences in copy number between two DNA samples, both of which arehybridized to the same hybridization area. It has advantages over CGH inthat it allows greater resolution to be achieved and has application tothe detection and diagnosis of genetic disorders induced by a change incopy number, in addition to other areas where copy number detection isimportant. While the particulars vary, a range of different probe typescan be used, including those encountered in oligonucleotide, PAC, andbacterial artificial chromosomes (BAC) arrays.

ArrayCGH is currently being used to support the efforts of clinicians inthe investigation of genomic imbalance in constitutional cytogeneticsand increasingly in oncology. These applications are incrediblydemanding such that the microarrays designed for these applications mustbe produced to far more rigorous standards than those used in academicor pre-clinical research applications.

ArrayCGH has an advantage over metaphase CGH in that the interpretationis much simpler and easily automated; in addition the time taken for thecomplete analysis is shorter. ArrayCGH can be used to detect aneuploidyin single cells and has been successfully applied to PGS. Single cellshave to be amplified for the technique and the same methods are employedas those used in metaphase CGH. ArrayCGH allows comprehensive analysisof the whole genome to be completed within 48 hours, which allowsaneuploidy screening without cryopreservation in PGS.

In order to achieve optimal assay results, arrayCGH requires the testand reference samples to be well matched in terms of quality andconcentration. In the context of PGS, the starting point for anyanalysis is the genetic material which is as representative as possibleof the fertilized embryo, or oocyte in the context of egg banking.Currently it is possible to examine the genetic material containedwithin a polar body or a blastomere, a single cell extracted from an 8cell embryo, or alternatively a small number of cells from a blastocystor associated biopsy. As only a limited amount of DNA can be obtainedfrom such material, most downstream analyses require DNA amplificationprocedures to be used in order to produce large numbers of copies of thestarting material. It is to be understood that polar bodies are ejectedas a fertilization process begins and there are two of them, PB1 andPB2. The process is not straight forward. Herein, the term “polar body”can comprise a body ejected or biopsied from a primary or secondaryoocyte.

While un-amplified genomic reference material may be used, correspondingarrayCGH results can show high noise levels due to poor matching ofamplified test with un-amplified reference. Thus, reference materialused in this context is often a ‘normal’ pooled DNA sample diluted tocontain a broadly similar quantity of DNA as a small number of singlecells. This diluted reference material is then amplified using the samemethod as the test sample. Even though these steps are taken to matchthe properties of the test and reference samples this is not alwayseffective and the clarity of results can vary. This may be for a widevariety of reasons, including: minor errors in the quantification of thestarting DNA and hence variable quantities of DNA in the diluted sample;variation due to the stochastic nature of the amplification process;amplification of impurities in the sample which are not present in thereference; low sample DNA “quality” leading to increased non-specificamplification; variability in the quantity and type of reagents used inthe extraction and storage of samples. In all cases, the resultantdifferences between amplification of sample and reference can both alterand obscure the results of the true amplification, leading to alteredarrayCGH profiles, and frequently increased noise and suppressed dynamicrange.

PGS is a diagnostic application, and it is standard practice for eachexperiment to include an internal control to demonstrate successfulfunctioning of the experiment, and also to assess variation in dynamicrange between experiments which, for example, may arise due to theamplification issues described previously. When using arrayCGH, the mostcommonly used approach to address this problem is to use a referencesample with known copy number gains/losses relative to the test sample.These can then be used as a measure of performance for each individualassay.

Most frequently, the reference sample is sex-mismatched against thetest, giving a shift on the log_(e) ratio of test over reference for theX and Y chromosomes, and consequently a measure of dynamic range. Whileapplicable in many contexts, in the case of PGS, however, it isgenerally not possible to know a priori the sex of the sample,especially in aneuploidy screening of blastomere or blastocyst biopsysamples that could be either sex. The use of a single reference asinternal control is therefore not reliably possible. Moreover, selectionof a single appropriate reference, with a known copy number imbalance inregions other than the sex chromosomes, to a test sample is generallynot possible, as the degree of copy number variation in embryos/oocytesis extremely high, and current research indicates that there are noregions which are predictably stable. In some embodiments, the selectionof an embryo for implantation can be made on the basis of aneuploidystatus. In other embodiments, selection is made on the basis of smallergenetic aberrations.

An alternative is to use a reference which includes non-human controlsequences. However, this approach is less than ideal as it is difficultto choose non-human sequences which accurately mimic the behavior ofhuman sequences. In any case, the use of non-human control sequences cansuffer from the same amplification biases, and other biases, and as suchchoice of a single reference can be challenging.

To overcome this problem in PGS, it would be necessary to carry out twoconventional arrayCGH hybridizations to analyze a single test sample,one against a male and another against a female reference to ensure thatthe assay is working correctly. However, the cost associated with thisapproach is unacceptably high for the application.

Where two or more cells are taken from an embryo, for example, from ablastocyst/trophoectoderm), the possibility of mosaicism in the testsample becomes significant in a PGS context, as embryos are frequentlymosaic. To complicate matters, the number of cells taken from the embryomay be unknown due to inaccuracy of biopsy methods. While arrayCGH candetect mosaicism, it provides no means to directly quantify thismosaicism due to a lack of sufficiently sophisticated internal controlsand furthermore, for the same reason, may mistake experimental noise formosaicism. ArrayCGH's reliance on a single reference sample is againproblematic in this context.

ArrayCGH requires contrasting fluorescent dyes to label the test andreference samples. The popular dye pair Cy3 and Cy5 is often used forarrayCGH. The Cy5 dye is susceptible to degradation by ozone in theenvironment and particularly when combined with high humidity, thisinfluence on assay quality can lead to the loss of experimental data.ArrayCGH is used wherein two fluorescently labeled samples arecompetitively hybridized to the same hybridization area, such thatthrough ratiometric comparison relative gain or loss of genetic materialcan be ascertained. Typically, one sample is a test sample of unknowngenetic make-up and one sample is a reference sample known to havenormal copy number, where normality is defined by the application inquestion. ArrayCGH is a powerful and robust technique, however the PGSapplication presents unique technical challenges. In some embodiments,the assessment of chromosomal content of an embryo, can be made eitherdirectly through taking cells after fertilization, or indirectly throughassessing polar bodies and thus the oocyte generating the embryo. Insome embodiments, an application exists whose only purpose is to assessthe content of the oocyte, and no embryo is necessarily generated. Thisis referred to herein as egg banking.

Buffart et al. (2008) suggest a modified arrayCGH technique that theyterm “across arrayCGH” (aaCGH), as an improvement to the currenttechnologies. AaCGH is similar to arrayCGH, but instead of hybridizationof the test and reference sample to a single hybridization area, testand reference samples are compared from separate hybridization areas.This method, independently developed by the authors of this patent,offers advantages in cost and potentially in data quality as it removesany noise due to dye bias. The quality of the profiles obtained usingaaCGH were reported to match or even surpass those obtained usingregular dual channel arrayCGH. The reference is described as beinghybridized at the same time, on the same slide, as the test using amulti format array, and the test and reference are labeled with the samefluorescent dye. They compare a single test sample with a singlereference sample. The method does not, however, overcome the uniquechallenges of PGS.

SNP array techniques, as distinct from arrayCGH, may also be used todetermine copy number in DNA samples, and have also been deployed forPGS applications. SNP arrays offer screening of all chromosomes andallow concurrent genotyping. The mechanism used is substantiallydifferent than the arrayCGH mechanism in that the technique is notcomparative. No reference sample is used and no co-hybridization isperformed, and the method for copy number assignment relies onquantification of individual alleles and subsequent ratiometric analysisin contrast to arrayCGH where individual alleles are not assessed.Disadvantages of SNP arrays include increased noise levels, longerprotocols, complexity of data interpretation and ethical implications,and possibly lower applicability to haploid samples.

The molecular cytogenetic technique of FISH (fluorescence in-situhybridization), which uses chromosome-specific DNA probes, hasfrequently been applied to PGS and gives detectable signals oninterphase nuclei. Although no amplification step is required, asignificant disadvantage exists is that only a limited number ofchromosomes can be assessed concurrently, limited by the number ofdistinct colors available for labeling of the DNA probes. The mostcomprehensive FISH methods used for routine embryo screening currentlyassess only half of the chromosomes, and thus, some chromosomalabnormalities are missed. Other disadvantages of FISH includeoverlapping signals which are difficult to score.

SUMMARY

A feature of the present teachings is to provide a method fordetermining the presence of a copy number imbalance in genomic DNA of atest DNA sample that reduces the risk of assay failure due to poormatching of test and reference samples associated with conventionalarrayCGH. This method can increase assay quality, accuracy, and yield.

Another feature of the present teachings is provide a method fordetermining the presence of a copy number imbalance in genomic DNA of atest DNA sample that separately measures hybridization of a single testsample to one hybridization array and hybridization of a set ofreference samples to one or more other hybridization arrays.

An additional feature of the present teachings is to provide a methodfor determining the presence of a copy number imbalance in genomic DNAthat comprises selection of a single optimal pairing of test DNA andreference DNA samples.

The present teachings provide a method for determining the presence of acopy number imbalance in genomic DNA of a test sample. The sample cancomprise labeling sample genomic DNA from a test sample, or anamplification product thereof, to form labeled test DNA, hybridizing thelabeled test DNA to a first hybridization array, labeling firstreference genomic DNA from a reference sample, or an amplificationproduct thereof, to form labeled first reference DNA, hybridizing thelabeled first reference DNA to a second hybridization array, labelingsecond reference genomic DNA from a second reference sample, or anamplification product thereof, to form labeled second reference DNA, andhybridizing the labeled second reference DNA to a third hybridizationarray. The method can comprise analyzing the first hybridization arrayafter the hybridizing of the labeled test DNA to determine signalintensities produced by hybridization of the labeled test DNA, analyzingthe second hybridization array after the hybridizing of the labeledfirst reference DNA to determine signal intensities produced byhybridization of the labeled first reference DNA, and analyzing thethird hybridization array after the hybridizing of the labeled secondreference DNA to determine signal intensities produced by hybridizationof the labeled second reference DNA.

The present teachings provide a method for estimating a copy number forat least one region of a sample genomic DNA by comparing the signalintensities of a test hybridization array with the signal intensity ofat least one of two or more reference hybridization arrays.

Additional features and advantages of the present teachings will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of thepresent teachings. The objectives and other advantages of the presentteachings will be realized and attained by means of the elements andcombinations particularly pointed out in the description and appendedclaims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide a further explanation of the presentteachings, as claimed.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate some of the embodiments of thepresent teachings and together with the description, serve to explainthe principles of the present teachings.

FIG. 1 is a flowchart depicting an exemplary method for determining copynumber of a test DNA sample, according to the present teachings.

FIG. 2 is a flowchart showing an exemplary method for preparing a set ofreference DNA samples, according to the present teachings.

FIG. 3 is a flowchart showing how copy number of a test DNA sample canbe determined, according to the present teachings.

FIG. 4 is a flowchart showing how regions of copy number change in atest DNA sample can be determined based on two ratio profiles, accordingto the present teachings.

FIG. 5 shows a pair of plots wherein the top plot shows a samplecompared to a male reference and the bottom plot shows a sample comparedto a female reference.

DETAILED DESCRIPTION

The present teachings relate to a method for detecting aneuploidy, orthe presence of smaller copy number imbalances or smaller imbalances ingenomic DNA. The method for detecting aneuploidy or detection of copynumber imbalance (“the detection method”) according to the presentteachings can be useful in analyzing an oocyte prior to egg banking orcan be useful in pre-implantation genetic screening (PGS) to identifythe number and complement of chromosomes within the cells of an embryoprior to implantation using in-vitro fertilization procedures. Thedetection method can identify chromosomal regions in genomic DNA that isrepresentative of the embryo for testing (“test DNA”), which contain anincreased or decreased copy number. The detection method can compriseusing single channel array comparative genomic hybridization (“singlechannel arrayCGH”), whereby a test DNA is hybridized to a hybridizationarray, and one or more DNA molecules for which the copy number isalready known (“reference DNA”) is hybridized to one or more differenthybridization arrays. A copy number imbalance in the test DNA can beidentified by detecting regions where the signal intensities resultingfrom hybridization of the reference DNA and hybridization of the testDNA, are different. Various features of the present teachings caninclude the genomic hybridization methods, devices, and kits describedin WO 96/17958 and in U.S. patent application Ser. No. 12/609,156, filedOct. 30, 2009, the contents of which are incorporated by referenceherein in their entireties.

The detection method can comprise labeling a test sample DNA obtainedfrom a test sample to form a test DNA and hybridizing the labeled testDNA to a first hybridization array. The test sample DNA can be labeledto permit detection and/or measurement of hybridization of the testsample DNA to the first hybridization array. The signals generated byhybridization of the labeled test DNA can be detected and analyzed todetermine signal intensity produced from the first hybridization array,and thereby obtain test hybridization results. The test hybridizationresults can be compared to reference hybridization results, or signalintensity generated by hybridization of one or more reference DNA to oneor more other hybridization arrays that are separate from the firsthybridization array. For example, the signal intensity for a referenceDNA can be determined by labeling a reference DNA and hybridizing thelabeled reference DNA to a second hybridization array. The signalsgenerated by hybridization of the labeled reference DNA can be detectedand analyzed to determine the signal intensity of the reference DNA. Thepresence of a copy number imbalance in the test DNA can be determined byidentifying one or more regions of the first hybridization array wherethe signal intensity differs from the signal intensity produced in oneor more corresponding regions of the second hybridization array.

The determination of a signal intensity produced by hybridization of thelabeled reference DNA can occur prior to or after the determination of asignal intensity produced by hybridization of the labeled test DNA. Ifthe determination of a signal intensity produced by hybridization of thelabeled reference DNA occurs prior to the determination of signalintensity produced by hybridization of the labeled test DNA, thereference hybridization results can be recorded and stored as historicalreference hybridization results. Test hybridization results which arelater obtained for a test sample DNA can then be compared to thehistorical reference hybridization results to determine a copy numberimbalance in the test DNA. Use of historical reference hybridizationresults can avoid the need to perform actual hybridizations for areference sample each time comparison with particular test hybridizationresults is desired.

More than one reference DNA or a plurality of reference DNA samples canbe used in the detection method. For example, after determining thesignal intensity of the reference DNA, the signal intensity for a secondreference DNA can be determined. The signal intensity for the secondreference DNA can be determined by labeling and hybridizing the secondreference DNA to a third hybridization array. The signals generated byhybridization of the labeled second reference DNA can be detected andanalyzed to determine the signal intensity produced by hybridization ofthe second reference DNA. The presence of a copy number imbalance can bedetermined by identifying one or more regions of the first hybridizationarray where the signal intensity differs from the signal intensityproduced in one or more corresponding regions of the second and/or thirdhybridization array.

The method for determining the presence of a copy number imbalance ingenomic DNA of a test sample can comprise labeling sample genomic DNAfrom a test sample, or an amplification product thereof, to form labeledtest DNA, hybridizing the labeled test DNA to a first hybridizationarray, labeling first reference genomic DNA from a reference sample, oran amplification product thereof, to form labeled first reference DNA,hybridizing the labeled first reference DNA to a second hybridizationarray, labeling second reference genomic DNA from a second referencesample, or an amplification product thereof, to form labeled secondreference DNA, and hybridizing the labeled second reference DNA to athird hybridization array. The method can comprise analyzing the firsthybridization array after the hybridizing of the labeled test DNA todetermine a signal intensity produced by hybridization of the labeledtest DNA, analyzing the second hybridization array after the hybridizingof the labeled first reference DNA to determine a signal intensityproduced by hybridization of the labeled first reference DNA, andanalyzing the third hybridization array after the hybridizing of thelabeled second reference DNA to determine a signal intensity produced byhybridization of the labeled second reference DNA. A copy number can beestimated for at least one region of the sample genomic DNA by comparingthe signal intensities of the first hybridization array with the signalintensity of at least one of the second hybridization array and thethird hybridization array.

The labeled first reference DNA can include at least one copy numberchange in one or more pre-defined regions of the genome, relative to thelabeled test DNA. The labeled second reference DNA can include at leastone pre-defined region which does not have the same copy number changerelative to the labeled test DNA, as the labeled first reference DNAdoes. In some cases, the signal intensity produced by hybridization ofthe labeled test DNA is compared to the signal intensity produced byhybridization of the labeled first reference DNA in the one or morepre-defined regions, the signal intensity produced by hybridization ofthe labeled test DNA is compared to the signal intensity produced byhybridization of the labeled second reference DNA in the one or morepre-defined regions, and the method further comprises determining adynamic range of the method based on an expected copy number. Thelabeled first reference DNA can be from a male animal of a firstspecies, for example, a mammal such as a human, and the labeled secondreference DNA can be from a female animal of the first species. Thelabeled first reference DNA and the labeled second reference DNA cancomprise a mixture of DNA from a male and from a female of a samespecies of animal. The labeled first reference DNA can include a trisomyand the second reference DNA can include a monosomy. In someembodiments, the first can include a small amplification on anychromosome, and the second can exclude such.

The signal intensity produced by hybridization of the labeled test DNAcan be compared to the signal intensity produced by hybridization of thelabeled first reference DNA in the one or more pre-defined regions, todetermine a first estimate of copy number, the signal intensity producedby hybridization of the labeled test DNA can be compared to the signalintensity produced by hybridization of the labeled second reference DNAin the one or more pre-defined regions, to determine a second estimateof copy number, and the first and second estimates of copy number can becombined to obtain an overall estimate of copy number. In someembodiments, the signal intensities are normalized before the copynumber is estimated.

In some cases, the first reference genomic DNA from a reference sample,or an amplification product thereof, can comprise an amplificationproduct produced by a first amplification technique, and the secondreference genomic DNA from a reference sample, or an amplificationproduct thereof, can comprise an amplification product produced by thesame first amplification technique. The first reference genomic DNA froma reference sample, or an amplification product thereof, can comprise aplurality of different amplification products each formed by amplifyinga different starting concentration of a same first reference genomicDNA. The method can comprise determining an aneuploidy status of a humanpolar body or embryo based on the copy number estimate. The method canfurther comprise using copy number information, for example, aneuploidystatus, to select embryos for implantation in IVF procedures. The methodcan comprise isolating genomic DNA from the test sample to form thesample genomic DNA or amplification product thereof.

The test sample can comprise at least one cell from an embryo. The firstgenomic reference DNA can comprise DNA obtained from tissue or cells ofan animal having a chromosomal anomaly. In some embodiments, the firstgenomic reference DNA comprises DNA obtained from mosaic tissue orcells. In some cases, the labeled first reference DNA has a firstconcentration of DNA and comprises normal male DNA, the labeled secondreference DNA has a second concentration of DNA and comprises the samenormal male DNA as the labeled first reference DNA, and the secondconcentration is diluted relative to the first concentration. Female DNAcan be used instead of male DNA, or in addition to male DNA. The labeledfirst reference DNA can comprise pooled genomic DNA extracted from bloodsamples taken from at least two individuals.

According to the present teachings, a method for determining thepresence of a copy number imbalance in genomic DNA of a test sample isprovided, comprising labeling a test DNA to form labeled test DNA,hybridizing the labeled test DNA to a first hybridization array,analyzing the first hybridization array after the hybridizing to obtainfirst hybridization results, and comparing the first hybridizationresults with historical reference hybridization results from thehybridization of a labeled first reference DNA to a second hybridizationarray. The method can further comprise comparing the first hybridizationresults with historical reference hybridization results from thehybridization of a labeled second reference DNA to a third hybridizationarray, and determining the presence of a copy number imbalance byidentifying one or more regions of the first hybridization array wherethe signal intensities differ from the signal intensities produced inone or more corresponding regions of at least one of the secondhybridization array and the third hybridization array. The labeled firstreference DNA can be from a male animal of a first species and thelabeled second reference DNA can be from a female animal of the firstspecies.

The present teachings also provide a library of reference array datasets stored in a processor. Each reference array data set can comprisedata gathered from a respective reference array during a copy numberhybridization assay carried out on the respective reference array,wherein (1) each reference array from which a respective data set isgathered, and includes elements which are common with each otherreference array from which a data set is gathered and (2) each copynumber hybridization assay, from which a respective data set isgathered, is carried out under one or more different conditions thaneach other copy number hybridization assay from which a data set isgathered. At least two reference array data sets of the library candiffer from each other. In some embodiments, some of the reference setsare generated under identical conditions, to assess variability in thetechnique. Each reference array data set can comprise fluorescent signalintensity data.

The present teachings also provide a method comprising comparing a testarray data set gathered from a test array during a copy numberhybridization assay, to the reference array data sets of the library,and using a signal processor to determine a ratio between a test arraydata set and a data set from the library. A best fit data set can bedetermined from the library and can be the reference array data setdetermined by the processor to maximize the SNR of the ratio set soobtained.

According to the present teachings, a kit is also provided, andcomprises a first copy number hybridization array, a second copy numberhybridization array identical to the first copy number hybridizationarray, a third copy number hybridization array identical to the firstcopy number hybridization array, a first reference genomic DNA, a secondreference genomic DNA, and instructions for comparing test resultsgenerated from a hybridization assay carried out on the first copynumber hybridization array, to test results generated from ahybridization assay carried out on the second copy number hybridizationarray using the first reference genomic DNA. The instructions can alsobe for comparing test results generated from a hybridization assaycarried out on the first copy number hybridization array, to testresults generated from a hybridization assay carried out on the thirdcopy number hybridization array using the second reference genomic DNA.In some cases, the first reference genomic DNA can comprise anamplification product of a reference genomic DNA. The present teachingsalso provide a kit comprising a copy number hybridization array, anelectronic storage medium comprising a plurality of reference array datasets stored thereon, and instructions for comparing a data setcorresponding to test results generated from a hybridization assaycarried out on the copy number hybridization array, to the plurality ofreference array data sets.

As the measurements of the test and reference DNA samples can occur inseparate hybridization arrays, contrasting dyes for labeling are notnecessary to carry out the detection method. Also, test DNA can becompared to an unlimited number of reference samples, rather than simplya co-hybridized reference. In this way as many comparisons as requiredcan be performed in order to determine an optimal assay result for thetest sample.

Use of more than one reference DNA can further avoid the risk of assayfailure due to poor matching of test and reference DNA and permitselection of a single optimal pairing of test and reference DNA. Theplurality of reference DNA can include reference DNA that iswell-matched to test DNA obtained from DNA amplification of a singlecell. In other words, a single reference DNA from the range of referenceDNA generated can be selected that gives the best comparison to the testDNA. For example, reference DNA well-matched to test DNA can be achievedby generating a range of reference DNA samples through smallmodifications in the amplification protocol. Such small modification inthe amplification protocol can lead to a spread of technical variation.In addition, the reference DNA that is generated can have specific knownbiological properties. For example, the reference DNA that is generatedcan be derived from a mosaic individual or from an individual of aparticular sex. The reference DNA that is generated can have one or morechromosomal anomalies. The reference DNA can be derived from acompromised cell in order to match the condition of the test sample. Thereference DNA can be derived from an individual biologically related tothe test sample.

Hybridization array, as used herein, can comprise a microarray, or acollection of solid support-bound unlabeled target nucleic acids(probes), for example, an array of clones which have been mapped tochromosomal locations. The hybridization array can comprise a pluralityof probes or target nucleic acid molecules, such as at least two targetnucleic acid molecules, bound to a solid support or surface. The targetnucleic acid molecules can be organized in predefined locations on thesolid surface with discrete locations for each of the probes. The targetnucleic acid molecules bound to the solid surface can be a plurality ofthe same target nucleic acid molecules, a plurality of different nucleicacid molecules, or a combination of the two. For example, in embodimentswhere it is desired to multiplex the detection assay (i.e., detect morethan one nucleic acid molecule at a time), a plurality of differenttarget nucleic acid molecules that bind to different nucleic acidmolecules can be used. The solid surface can be any surface suitable forarray CGH including both flexible and rigid surfaces. Flexible surfacescan include, but are not limited to, nylon membranes. Rigid surfaces caninclude, but are not limited to, glass slides. The solid surface canfurther comprise a three dimensional matrix or a plurality of beads. Anysuitable method for immobilizing the target nucleic acids on the solidsurface can be used.

It should be understood that while hybridization of DNA is describedherein, any kind of nucleic acid, such as RNA, DNA, or cDNA, can beused. Similarly, the target nucleic acid molecules or probes can be, forexample, RNA, DNA, or cDNA. The nucleic acids can be derived from anyorganism. The probes can be synthetic oligonucleotides or can be derivedfrom cloned DNA or PCR products. The oligonucleotides can be synthesizedin situ or synthesized and then arrayed ex situ. The cloned DNA can bebacterial artificial chromosome (BAC) clones or PI-derived artificialchromosomes (PAC). The sequence of the nucleic acid molecules canoriginate from a chromosomal location known to be associated withdisease, can be selected to be representative of a chromosomal regionwhose association with disease is to be tested, or can correspond togenes whose transcription is to be assayed.

A reference DNA can be labeled and hybridized to a hybridization array.The hybridization array can be washed to remove any non-specificallybound labeled material. The hybridization array can then be scanned andthe signal intensity of the reference DNA can be recorded and stored ashistorical reference hybridization results for subsequent comparisonwith test hybridizations results for a test DNA sample. Similarly, a setof historical reference hybridization results can be generated andrecorded for a plurality of different reference DNA. The plurality ofreference DNA samples can be labeled and hybridized individually toseparate hybridization arrays having the same array design. Thehybridization arrays can be scanned and the scanned data can betransformed into historical reference hybridization results, and storedfor later use. Since the historical reference hybridization results canbe recorded, hybridization of the plurality of reference DNA does notneed to be done more than once. The historical reference hybridizationresults can be used repeatedly for subsequent assays using one or moredifferent test DNA. The test DNA can be labeled and hybridized to ahybridization array having the same array design as the hybridizationarrays used to obtain the historical reference hybridization results.The historical reference hybridization results can be transferredelectronically or in an electronic storage medium to end users.

It should be understood that “scanning” as used herein, refers to anyconventional method carried out by a scanner that would allow detectionof hybridization of a sample to a hybridization array. Scanning caninclude, for example, emitting light from a light source of a thescanner and, at a detector of the scanner, receiving the emitted lightthat reflects off of a respective location of the hybridization array.In some embodiments, scanning can include, for example, excitingfluorescent dyes on a microarray, and at a detector of the scanner,measuring emitted fluorescent intensity. Scanning is further describedfor example, in WO 96/17958 and in U.S. patent application Ser. No.12/609,156, filed Oct. 30, 2009, each of which is incorporated herein inits entirety by reference.

FIG. 1 is a flowchart depicting one method for determining copy numberof a test DNA sample. As shown in FIG. 1, a labeled test DNA sample canbe hybridized to hybridization area A. The signal intensity or amplitudegenerated by the hybridization can be measured to construct a test DNAsample amplitude profile. The test DNA sample amplitude profile can benormalized. A set of reference DNA samples can be selected andseparately hybridized to hybridization areas other than hybridizationarea A. The signal intensity or amplitude generated by the hybridizationof each reference DNA can be measured to construct a reference DNAamplitude profile. The reference DNA amplitude profile can benormalized. Copy number for the test DNA sample can be determined bycomparing the test DNA sample amplitude profile to the reference DNAamplitude profile. In some embodiments, by identical array, what ismeant is an array comprising a lot of the same content, for example, atleast 90% of the same content but which can differ in other content.Similarly, slight variations in amplification procedure can also be usedwhile still being considered identical.

In some embodiments, each reference DNA will have an associatedamplitude profile. An initial estimate of copy number is then determinedby taking the ratio of the test amplitude to the reference amplitude (ormultiple reference amplitudes), and possibly various normalizations.There is naturally noise in this estimate of copy number, and a furtherstep can be used to assess whether an estimated copy number is likely tocorrespond to a genuine change in biological copy number, or is simplydue to noise (and therefore a copy number of zero).

The plurality of different reference DNA can comprise reference DNA thathave specific known biological properties. For example, the referenceDNA can be obtained from male or female samples. The reference DNA canbe obtained from cell lines with a desired chromosomal anomaly. Thereference DNA can be obtained from mosaic sample. Synthetic mosaicreference samples can be constructed by combining cells or extracted DNAwith differing but known karyotypes so as to replicate mosaic karyotypepatterns. This combining can occur at any stage during the preparationof reference DNA, or following labelling.

The reference DNA can be derived from a compromised cell in order tomatch the condition of the test sample. Reference DNA can be derivedfrom individuals biologically related to the individual from whom thetest sample was taken.

The test DNA can be prepared from a test sample, such as, a test cell,cell population, or tissue under study. The test DNA can be isolatedfrom one or more test cells. The test DNA can be obtained from a polarbody wherein half of an egg's chromosome complement is ejected prior tofertilization. The test DNA can be obtained from a blastomere, a singlecell extracted from an eight cell embryo, or a small number of cellsfrom a blastocyst or associated biopsy, for example, a trophoectoduralbiopsy. The test cell can comprise at least one cell from an embryo. DNAamplification procedures can be used in order to produce large numbersof copies of the test DNA.

The reference DNA can be prepared from a reference cell, cellpopulation, or tissue. Reference cells can be normal non-diseased cells,or they can be from a sample of diseased tissue that serves as astandard for other aspects of the disease. The reference DNA is thegenomic material for which the copy number of the genes or nucleic acidmolecules of interest are already known.

The reference DNA can be generated using a variety of startingmaterials. Examples of starting materials can include tissue, such asblood, donated by one or more individuals. Other sources of startingmaterials can include single cells. Standard procedures can be used toisolate the reference DNA from appropriate tissues or cells. Thereference DNA or starting material can be chosen from an individualhaving normal chromosomes and/or an individual having chromosomalanomalies, such as gain or loss of one or more chromosomes oralternatively gain or loss of one or more chromatid. The single cellscan be derived from cell culture in vitro or can be ex vivo human cells,either of the same type as the intended test sample or of a differenttype. Single cells can be selected because they have a chromatinstructure which is similar or dissimilar to that of the intended testsample, for example sperm cells with dense chromatin can be selected.Similarly cells can be chosen which are at differing stages of the cellcycle. Alternatively, high quality and concentrated genomic referenceDNA can be extracted from cell culture or blood and can be dilutedpost-extraction to levels that are comparable to concentrations obtainedfrom a single cell.

Although, a familial relationship between the donor of materials used toproduce reference DNA and the test sample can be present, such arelationship is not required. Reference material can be obtained fromone or both parents in order to be able to make direct comparisonsbetween the test sample and parental samples.

A variety of conditions can be used to generate a plurality of referenceDNA of differing quality. Reference DNA can be generated from cells ofdiffering integrity, for example, reference samples generated fromcompromised cells. Reference DNA can be treated post-sample collection,such as DNA extracted from formalin-fixed paraffin-embedded tissue. Thereference DNA can be subject to physical treatments, such as heating orsonication. Chemical treatments, such as, enzymatic digestion orproteinase digestion can also be used. Other treatments can beperformed, which simulate test sample conditions during IVF procedures.Such treatments can include mineral oil contamination and contaminationwith culture media in order to normalize any contribution these factorsmake to differences in assay performance between test and referencesamples.

The preparation of reference DNA can involve the application of wholegenome amplification. The whole genome amplification protocol can bevaried so as to introduce variations into the amplified DNA products.For amplification, a SUREPLEX DNA amplification, or other suitableamplification can used. The precise nature of the DNA amplification usedis not critical to the teachings. While un-amplified genomic referenceDNA can be used, high noise levels can result due to poor matching ofamplified test DNA with un-amplified reference DNA. Thus, the referencematerial used can be a ‘normal’ pooled DNA sample diluted to contain abroadly similar quantity of DNA as a small number of single cells. Thisdiluted reference material can then be amplified using the same methodas the test sample. In order to compensate for differences betweenamplification of test and reference DNA, the set of reference samplescan be carefully constructed so as to span the space of variationsresponsible for poor matching. This strategy can reduce the risk ofassay failure due to poor matching of test and reference samplesassociated with conventional arrayCGH. Separating the measurement oftest and reference samples, as described herein, allows comparison ofmeasurements made on a single test sample with measurements made on aseries of reference samples. In this way a single optimal pairing oftest and reference samples can be found, or alternatively, results frommultiple comparisons can be combined and compared to test DNA.

FIG. 2 is a flowchart showing an exemplary method for preparing a set ofreference DNA samples. As shown in FIG. 2, variations can be introducedto reference DNA by varying the amplification protocol that is used tocreate a set of reference DNA samples. A number of identical referenceDNA sample pairs can be constructed. Each reference DNA sample pair cancomprise a normal male reference DNA and a normal female reference DNA.Each sample pair can be diluted to a different extent to create a serialdilution. Each reference DNA with a pair can be diluted to the sameextent. Each reference DNA of each sample pair can be amplifiedseparately using an amplification method that is the same as that usedto amplify a test DNA.

The test DNA and the reference DNA can be labeled to allow detection ofhybridization complexes. The particular label attached to the DNA is nota critical aspect of the teachings, as long as the label does notsignificantly interfere with the hybridization of the DNA to the targetnucleic acid molecules. The label can be any material having adetectable physical or chemical property. The label can include, forexample, a fluorescent dye, a radiolabel, or an enzyme. Generally,fluorescent labels commonly used for arrayCGH, such as Cy3 and Cy5, arepreferred. A CYTOCHIP labeling kit from BLUEGNOME, for example, can beused. Standard methods for detection and analysis of signals generatedby the labels can be used. For fluorescent labels, standard methodsgenerally used in array comparative genomic hybridization (“arrayCGH”)can be used. The hybridization arrays can be imaged in a fluorescencemicroscope with a polychromatic beam-splitter. The different colorimages can be acquired with a CCD camera, a laser scanner, a combinationthereof, and the like, and the digitized images can be stored in acomputer. A computer program can then be used to analyze the signalsproduced by the array.

The selection of a single optimal pairing of test and reference DNA anddetermination of a copy number imbalance in test DNA can be automated tosimplify data analysis and interpretation and/or increasereproducibility. A set of algorithms can be provided which automate thechoice of reference data as well as scoring of the assay. Thesealgorithms can simplify data analysis, interpretation, and/or increasereproducibility. The algorithms can comprise a reference selectionalgorithm and a calling algorithm. The reference selection algorithm cancompare the test hybridization results for a test DNA with a set ofcorresponding historical reference hybridization results and determinewhich reference DNA of the plurality of reference DNA yields the bestcomparison to the test DNA.

In the event that the detection method can suffer from spatial noise dueto inter array hybridization variability and/or hybridizing samples ondifferent days. As such, the detection method can comprise methods forspatial bias correction or methods to spatially correct for inter-arrayhybridization. Any spatial bias which may exist due to differences inhybridization of the test DNA and the reference DNA can be detected andremoved by methods known in the art, for example, as described in U.S.patent application Ser. No. 12/609,156, filed Oct. 30, 2009, thecontents of which are incorporated by reference herein in theirentireties.

The reference selection algorithm can characterize the results of eachtest/reference comparison with a performance metric. The performancemetric can be, for example, signal to noise ratio. The signal componentcan be defined as the difference between the medians of the log₂ ratiosof a chosen chromosome pair of the hybridization array where ratios arebetween test and reference. The noise component can be obtained bytaking the set of target nucleic acids or probes in the hybridizationarray for each chromosome and subtracting the chromosome median log₂ratio from each individual probe log₂ ratio. Once the chromosome trendsare removed, the noise can be determined by calculating the interquartile range over all probes.

The reference selection algorithm can select the reference DNA thatmaximizes the SNR of the ratiometric data, indicative of copy number inthe test DNA. The test-reference pairing can then be automaticallypresented to the calling algorithm. The calling algorithm can be appliedto identify regions of copy number imbalance between the test andreference samples. The calling algorithm can compare the observedpattern of imbalance to the expected pattern of imbalance. Because thekaryotype of the reference sample is known, the karyotype of the testsample can then be inferred. A final classification of the sample can beeither “euploid” (no copy number imbalance) or “aneuploid” (copy numberimbalance). In some cases, the test data can be of poor quality, suchthat any results obtained would be unreliable. In these circumstances,the calling algorithm may classify the result as “no result.”

FIG. 3 is a flowchart showing how copy number of a test DNA sample canbe determined. As shown in FIG. 3, a set of virtual ratio profiles canbe constructed by dividing a test DNA sample amplitude profile by thereference DNA amplitude profile of each reference DNA. The “noise” and“dynamic range” of each virtual ratio profile can be calculated. Thedynamic range can be calculated on a basis of ratio of X/Y chromosomes.A pair of ratio profiles can be selected, corresponding to a referencesample pair, which has the best combination of low noise and expecteddynamic range. In other words, the best “amplification” match of testDNA and reference DNA pair can be selected. The calling algorithm can beemployed to determine regions of copy number change in the test DNAbased on the pair of ratio profiles.

It should be understood that if the test sample from which the test DNAis isolated is from a first polar body, one optimal pairing of referenceDNA and test DNA can be made although it may still be desirable toselect the second for reasons elsewhere outlined. If the test DNA isisolated from a blastomere biopsy for which the gender of the sample isnot known in advance, the reference selection algorithm can select twooptimal pairings. In some embodiments, an unlimited number of pairingscan be chosen. If the test DNA is isolated from a blastomere biopsy, thereference DNA can comprise male genomic DNA and female genomic DNA ofvarying quality. The two optimal pairings can comprise a test DNA with amale reference DNA and the test DNA with a female reference DNA. Thecalling algorithm can then identify copy number imbalances present inone or both of the pairings. These imbalances can be compared to theexpected pattern of imbalances. Because the karyotypes of both referencesamples are known, the karyotype of the test sample can then beinferred.

FIG. 4 is a flowchart showing how regions of copy number change in atest DNA sample on the basis of two ratio profiles can be determined. Asshown in FIG. 4, the signal intensity after hybridization of a normaltest DNA sample can be compared to the signal intensity of the normalmale and female reference DNA to determine a copy number imbalance. Thetest DNA sample can be the same sex of the reference DNA that has thesame copy number as the test DNA. Ratio profiles of a normal test DNAand a normal male reference DNA as well as the test DNA and a normalfemale reference DNA can be obtained. For each ratio profile, analgorithm can be used to determine aberrant regions of potentialsignificance in the test DNA, in other words, aberrant regions which aresignificantly larger than baseline noise in the profile. Significantratio levels consistent with genuine copy number change can bedetermined by considering the X/Y ratios in the ratio profilecorresponding to the test DNA sample and its sex-mismatched referenceDNA. A determination can be made whether each aberrant region isconsistent with a genuine copy number change or not, using significantratio levels obtained at previous stages. Copy number calls fromindividual ratio profiles can be combined to form a single copy numbercall for the test DNA, for example, by averaging. It should beunderstood, however, that X and Y chromosome region calling canpreferentially be on the basis of the ratio profile consistent with asex-match between sample and reference.

More than one test DNA can be hybridized to the same hybridization area.For example, two or more test DNA samples can be labeled with differentdyes and hybridized to the same hybridization area.

In some embodiments, the test sample is male, the first reference isfemale, and the second reference is male. In some cases, the test sampleis female, the first reference is male, and the second reference isfemale. In either case, the first reference can be used to establish adynamic range which can be used to detect all the chromosomes, and thesecond reference can be used for calling on X/Y. To address challengesproduced by not knowing the sex of the sample, if the test sample is apolar body a female reference and a male reference can both be used suchthat one reference is matched to the sample and the other providesdynamic range information.

It is to be understood that “copy number” as used herein is relative toa reference genome. For example, if a reference is a mixture of male DNAand female DNA, copy number is not necessarily an integer. In some casesa triploid reference can be used as a normal and would have yet adifferent copy number.

According to the present teachings, a first reference is anamplification product of a sample including a copy number change in afirst determined reference. In some embodiments, a first reference is anamplification product of a first predetermined region and a secondreference has a deletion in the predetermined region rather than nochange in the predetermined region.

According to the present teachings, first, second, third, and any otherhybridization areas can be the same if different labels are used.

While the methods described herein are designed for IVF, the methods canalso be applicable in a prenatal, oncology, and/or stem cell context.The detection method further allows a multitude of referencesrepresenting different degrees of mosaicism, under differentamplification conditions to be used.

Even where the sex of the sample is known, it is still generally usefulto be able to run the sample against both a sex matched and sexmismatched references. For example, in the case of testing a polar body,it can be known that the sample is female and therefore a male referenceis the defacto choice for reference in order to get an internal dynamicrange control. However, this can have the side effect of complicatinginterpretation of the X and Y chromosomes of the polar body. It istherefore still an advantage wherein the sex mismatch is used tocalculate dynamic range and provide calls on chromosomes 1-22, and thesex match is used to call all chromosomes.

In some cases, in predefined regions, at least one reference sample hascopy number difference with the test sample. This copy number differencecan be used as a “control” to effectively indicate the dynamic range ofthe experiment (which is dependent on the individual test sample, andhybridization conditions, in question). For example, if a copy numberdifference of “1” is expected between the test and a particularpredefined region in a reference, and due to experimental reasons, thedifference is only “0.25,” information is gained about the dynamic rangeof the experiment such that 0.25 can be significant in a particularexperiment. This estimate of dynamic range, or significance,” can thenbe used to assess copy number changes in other reference datasets, ordifferent chromosomes on the same reference dataset.

EXAMPLES

A sample comprising a normal female missing 1 copy of chromosomes 13 and19 was provided. FIG. 5 shows a pair of plots wherein the top plot ofeach shows a sample compared to a male reference and the bottom plot ofeach shows a sample compared to a female reference. In the top plot ofFIG. 5, the sample is compared with a male reference and the Xchromosome shows as a gain because there are two copies of X in thefemale and only one in the male reference, and similarly the Ychromosome appears as a loss as there is no Y in the female and one inthe male reference. These expected X/Y changes provide an indication ofwhat is significant. Chromosomes 13 and 19 are clear losses.

The bottom plot in FIG. 5 shows the same sample compared with a femalereference. In this case, the number of X and Y chromosomes is expectedto be the same in both sample and reference. Once again, the losses onchromosome 13 and 19 are visible, particularly when combined with theinformation about dynamic range obtained from the X/Y comparisons in thetop plot.

The entire contents of all cited references in this disclosure areincorporated herein in their entireties by reference. Further, when anamount, concentration, or other value or parameter is given as either arange, preferred range, or a list of upper preferable values and lowerpreferable values, this is to be understood as specifically disclosingall ranges formed from any pair of any upper range limit or preferredvalue and any lower range limit or preferred value, regardless ofwhether ranges are separately disclosed. Where a range of numericalvalues is recited herein, unless otherwise stated, the range is intendedto include the endpoints thereof, and all integers and fractions withinthe range. It is not intended that the scope of the teachings be limitedto the specific values recited when defining a range.

Other embodiments of the present teachings will be apparent to thoseskilled in the art from consideration of the present specification andpractice of the present teachings disclosed herein. It is intended thatthe present specification and examples be considered as exemplary onlywith a true scope and spirit of the teachings being indicated by thefollowing claims and equivalents thereof.

1. A method for determining the presence of a copy number imbalance ingenomic DNA of a test sample, comprising: a) labeling sample genomic DNAfrom a test sample, or an amplification product thereof, to form labeledtest DNA; b) hybridizing the labeled test DNA to a first hybridizationarray; c) labeling first reference genomic DNA from a reference sample,or an amplification product thereof, to form labeled first referenceDNA; d) hybridizing the labeled first reference DNA to a secondhybridization array; e) labeling second reference genomic DNA from asecond reference sample, or an amplification product thereof, to formlabeled second reference DNA; f) hybridizing the labeled secondreference DNA to a third hybridization array; g) analyzing the firsthybridization array after the hybridizing of the labeled test DNA todetermine signal intensities produced by hybridization of the labeledtest DNA; h) analyzing the second hybridization array after thehybridizing of the labeled first reference DNA to determine signalintensities produced by hybridization of the labeled first referenceDNA; i) analyzing the third hybridization array after the hybridizing ofthe labeled second reference DNA to determine signal intensitiesproduced by hybridization of the labeled second reference DNA; and j)estimating the copy number of at least one region of the sample genomicDNA by comparing the signal intensities of the first hybridization arraywith the signal intensities of at least one of the second hybridizationarray and the third hybridization array.
 2. The method of claim 1,wherein the labeled first reference DNA includes at least one copynumber change in one or more pre-defined regions of the genome, relativeto the labeled test DNA.
 3. The method of claim 2, wherein the labeledsecond reference DNA includes at least one pre-defined region which doesnot have the same copy number change relative to the labeled test DNA,as the labeled first reference DNA does.
 4. The method of claim 2,wherein the signal intensity produced by hybridization of the labeledtest DNA is compared to the signal intensity produced by hybridizationof the labeled first reference DNA in the one or more pre-definedregions, the signal intensity produced by hybridization of the labeledtest DNA is compared to the signal intensity produced by hybridizationof the labeled second reference DNA in the one or more pre-definedregions, and the method further comprises determining a dynamic range ofthe method based on an expected copy number.
 5. The method of claim 1,wherein the labeled first reference DNA is from a male animal of a firstspecies and the labeled second reference DNA is from a female animal ofthe first species.
 6. The method of claim 1, wherein the labeled firstreference DNA and the labeled second reference DNA comprises a mixtureof DNA from a male and from a female of a same species of animal.
 7. Themethod of claim 1, wherein the signal intensity produced byhybridization of the labeled test DNA is compared to the signalintensity produced by hybridization of the labeled first reference DNAin the one or more pre-defined regions, to determine a first estimate ofcopy number, the signal intensity produced by hybridization of thelabeled test DNA is compared to the signal intensity produced byhybridization of the labeled second reference DNA in the one or morepre-defined regions, to determine a second estimate of copy number, andthe first and second estimates of copy number are combined to obtain anoverall estimate of copy number.
 8. The method of claim 1, whereinsignal intensities are normalized before the copy number is estimated.9. The method of claim 1, wherein the first reference genomic DNA from areference sample, or an amplification product thereof, comprises anamplification product produced by a first amplification technique, andthe second reference genomic DNA from a reference sample, or anamplification product thereof, comprises an amplification productproduced by the same first amplification technique.
 10. The method ofclaim 1, wherein the first reference genomic DNA from a referencesample, or an amplification product thereof, comprises a plurality ofdifferent references at different respective concentrations.
 11. Themethod of claim 1, further comprising: hybridizing a labeled thirdreference DNA to a fourth hybridization array; hybridizing a labeledfourth reference DNA to a fifth hybridization array; hybridizing alabeled fifth reference DNA to a sixth hybridization array; wherein theestimating the copy number comprises comparing the signal intensities ofthe first hybridization array with signal intensities generated by eachof the second, third, fourth, fifth, and sixth hybridization arrays. 12.The method of claim 1, further comprising determining an aneuploidystatus of a human polar body or embryo based on the copy numberestimate.
 13. The method of claim 12, further comprising implanting anembryo based on the aneuploidy status determined in an IVF procedure.14. The method of claim 1, further comprising isolating genomic DNA fromthe test sample to form the sample genomic DNA or amplification productthereof.
 15. The method of claim 1, wherein the test sample comprises atleast one cell from an embryo or associated biopsy.
 16. The method ofclaim 1, wherein the first genomic reference DNA comprises DNA obtainedfrom tissue or cells of an animal having a chromosomal anomaly.
 17. Themethod of claim 1, wherein the first genomic reference DNA comprises DNAobtained from mosaic tissue or cells.
 18. The method of claim 1, whereinthe labeled first reference DNA has a first concentration of DNA andcomprises an amplification product of a first concentration of a firstDNA, and the labeled second reference DNA comprises an amplificationproduct of the first DNA produced after diluting the first concentrationof first DNA. 19-21. (canceled)
 22. A library of reference array datasets stored in a processor, each reference array data set comprisingdata gathered from a respective reference array during a copy numberhybridization assay carried out on the respective reference array,wherein (a) each reference array from which a respective data set isgathered, is substantially identical or identical to each otherreference array from which a data set is gathered; (b) each copy numberhybridization assay, from which a respective data set is gathered, iscarried out under the same or one or more different conditions than eachother copy number hybridization assay from which a data set is gathered;and (c) at least two reference array data sets of the library differfrom each other. 23-26. (canceled)
 27. A kit comprising: a first copynumber hybridization array; a second copy number hybridization arrayidentical to the first copy number hybridization array; a third copynumber hybridization array identical to the first copy numberhybridization array; a first reference genomic DNA; a second referencegenomic DNA; and instructions for (a) comparing test results generatedfrom a hybridization assay carried out on the first copy numberhybridization array, to test results generated from a hybridizationassay carried out on the second copy number hybridization array usingthe first reference genomic DNA, and for (b) comparing test resultsgenerated from a hybridization assay carried out on the first copynumber hybridization array, to test results generated from ahybridization assay carried out on the third copy number hybridizationarray using the second reference genomic DNA. 28-30. (canceled)